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RESEARCH ARTICLE

Parasite Specific Antibody Increase Induced by an Episode of Acute P. falciparum Uncomplicated Malaria Mark Kaddumukasa1*, Catherine Lwanira2, Allan Lugaajju2, Elly Katabira1, Kristina E. M. Persson3,4, Mats Wahlgren3, Fred Kironde2,5

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1 Department of Medicine, College of Health Sciences, Makerere University, Kampala, Uganda, 2 College of Health Sciences, Makerere University, Kampala, Uganda, 3 Department of Microbiology, Tumour and Cell Biology, Karolinska Institutet, Stockholm, Sweden, 4 Department of Laboratory Medicine, Lund University, Stockholm, Sweden, 5 Habib Medical School, IUIU, Kampala, Uganda * [email protected]

Abstract OPEN ACCESS Citation: Kaddumukasa M, Lwanira C, Lugaajju A, Katabira E, Persson KEM, Wahlgren M, et al. (2015) Parasite Specific Antibody Increase Induced by an Episode of Acute P. falciparum Uncomplicated Malaria. PLoS ONE 10(4): e0124297. doi:10.1371/ journal.pone.0124297 Academic Editor: Érika Martins Braga, Universidade Federal de Minas Gerais, BRAZIL Received: October 23, 2014 Accepted: March 11, 2015 Published: April 23, 2015 Copyright: © 2015 Kaddumukasa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This research was conducted with funding from the European & Developing Countries Clinical Trials Partnership (EDCTP) grant (number IP.2007.31100.001) and from Makerere Sida-SAREC collaborative grant for research and training. The research leading to these results has also received funding (to FK and MW) from the European Community's Seventh Framework Programme (FP7/ 2007-2013) under grant agreement N° 242095 (www. evimalar.org). The funders had no role in study

Introduction There is no approved vaccine for malaria, and precisely how human antibody responses to malaria parasite components and potential vaccine molecules are developed and maintained remains poorly defined. In this study, antibody anamnestic or memory response elicited by a single episode of P. falciparum infection was investigated.

Methods This study involved 362 malaria patients aged between 6 months to 60 years, of whom 19% were early-diagnosed people living with HIV/AIDS (PLWHA). On the day malaria was diagnosed and 42 days later, blood specimens were collected. Parasite density, CD4+ cells, and antibodies specific to synthetic peptides representing antigenic regions of the P. falciparum proteins GLURP, MSP3 and HRPII were measured.

Results On the day of malaria diagnosis, Immunoglobulin (IgG) antibodies against GLURP, MSP3 and HRP II peptides were present in the blood of 75%, 41% and 60% of patients, respectively. 42 days later, the majority of patients had boosted their serum IgG antibody more than 1.2 fold. The increase in level of IgG antibody against the peptides was not affected by parasite density at diagnosis. The median CD4+ cell counts of PLWHAs and HIV negative individuals were not statistically different, and median post-infection increases in anti-peptide IgG were similar in both groups of patients.

Conclusion In the majority (70%) of individuals, an infection of P. falciparum elicits at least 20% increase in level of anti-parasite IgG. This boost in anti-P. falciparum IgG is not affected by parasite density on the day of malaria diagnosis, or by HIV status.

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design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Introduction Plasmodium falciparum malaria is a leading cause of morbidity and mortality among children in sub-Saharan Africa [1]. Humoral immune responses are believed to be critical to effective immunity against Plasmodium falciparum [2] and play a key role in the development of naturally acquired immunity to malaria [3, 4]. However, protective immunity to malaria takes years to develop and requires repeated infections [5]. Earlier studies showed that transfer of serum or antibody preparations from partially immune residents of endemic regions to non-immune individuals has beneficial anti-malarial activity [6, 7] and that malaria specific antibodies are associated with protection against malaria [8, 9]. In general, high antibody levels are associated with reduced susceptibility to clinical malaria [10, 11]. However, how the malarial antibodies are acquired, maintained, and boosted by regular parasite infections in endemic areas is little understood. Yet, if natural boosting of anti-malaria immunity is well elucidated, the information can be of great value in developing robust vaccines against malaria. Earlier studies have shown that, when individuals are exposed to a new malaria infection, parasite-specific antibody levels usually increase markedly within 1 or 2 weeks after the onset of symptoms [12, 13]. The boosted antibodies normally decline fast after an infection is resolved, suggesting that protective memory for specific antibody response is not efficiently formed or is functionally impaired [14]. However, moderate antibody levels persist in many individuals. Such persistence has been shown to increase with age among young children [15]. Nevertheless, little is known about how ordinary P. falciparum infections work to build antibody responses to candidate vaccine molecules. We know very little about the extent to which natural infections will boost antibodies generated by candidate malaria vaccines. Candidate blood-stage vaccines include GMZ2 which has been tested in children and adults [16, 17] and comprises glutamine rich protein (GLURP) and merozoite surface protein 3 (MSP3) [9, 14, 18]. MSP3 and GLURP are believed to be involved in erythrocyte invasion [19, 20] and are important vaccine candidates [21, 22]. Indeed, high levels of specific cytophilic antibodies against MSP3 and GLURP have been reported to be associated with protection against clinical malaria [23, 24]. A few clinical trials where whole sporozoite or trophozoite forms were used as vaccine showed that development of host protective immunity against malaria can be achieved although such vaccination would require large numbers of parasites [25, 26]. To gain further insight into the boosting of parasite specific antibody response following acute P. falciparum uncomplicated malaria, we investigated the antibody anamnestic response against three synthetic peptides [22] representing MSP3, GLURP, and Histidine Rich Peptide— II (HRPII) as well as parasitized erythrocyte (PE) lysate antigens. For this, we identified bloodsmear positive P. falciparum infected patients on day 0 (baseline), and collected venous blood for determination of parasite density, CD4+ cell counts and sero-prevalence of antibodies aganist the synthetic peptides and to antigens in PE lysate. The patients were treated with artemether—lumefantrine (co-artem) according to standard treatment guidelines. At day 42, blood was collected from the convalescent patients to determine post-infection levels of specific Immunoglobulin (IgG) and sero-prevalence to the three peptides. The relationship between the levels of anti-peptide antibodies for day 0 and day 42 was assumed to represent a reasonable measure of secondary anti-P. falciparum antibody boost or increase after an acute episode of uncomplicated malaria.

Materials and Methods Study area and subject enrolment The study was conducted at Kasangati Health Centre (KHC) which is about 20 km, north east of Kampala the capital city of Uganda. Patients were recruited from the peri-urban villages

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located within 10 km of KHC. Kasangati lies within a moderate transmission area, with a peak transmission after the two main rainy seasons (February-March and September-October) every year. This study was conducted from November 2010 to January 2011 after the September—October rains. Consecutive sampling was used to enrol a prospective cohort of participants. Eligibility criteria included: uncomplicated malaria as defined previously [27]. In brief, inclusion criteria were history of fever in the previous 24 hr, axillary temperature  37.5°C, haemoglobin > 5 g/dl, P. falciparum mono-infection at any parasitemia, and informed written consent from participants or their parents or guardians for minors, agreement to come to the study clinic for 42 day follow up, age: more than 6 months. A detailed clinical examination was performed and data entered into a pre-tested study questionnaire. The first dose of Artemether-Lumefantrine was administered under direct observation to ensure compliance. If a patient vomited within 30 min, the dose was repeated. The remaining doses were administered at home unsupervised. Human subject protection and ethics approval statement. A written informed consent was sought from all study participants before they were enrolled into the study. The study was approved by the Makerere University, School of Medicine, Research and Ethics Committee (SOMREC) and the Uganda National Council of Science and Technology (UNCST), 2007– 045.

Clinical investigations and follow-up On the day of recruitment, a finger prick blood specimen was taken and thick smears prepared and treated with Giemsa stain. Participants found to be smear positive for P. falciparum monoinfection were subsequently enrolled and a venous blood sample taken for sera and estimation of CD4+ cells. Follow-up of patients was arranged for day 42 and participants were asked to return to the clinic whenever they felt unwell within the subsequent 6 weeks. Follow-up consisted of history taking, a physical examination and blood smear examination. During the day 42 visit, a blood specimen was collected to re-assess the antibody levels. Standardized treatment was provided according to the national treatment guidelines. Patients who failed to return on day 42 were visited at their homes by our social worker and brought to the health unit for examination and medical care. Day 42 was selected as suitable for assessing the secondary antibody response after P. falciparum infection [28] and determining clinical outcome according to the WHO treatment guidelines [29, 30].

CD4 + T cell counts and examination of stained blood smears for parasites Thick smears were stained with 10% Giemsa stain for 10 min, sexual and asexual parasites were counted against every 200 white blood cells (WBCs). Results were multiplied by 40 to determine the parasite density (parasites/μL), assuming a normal WBC count of 8000 per μl [31, 32]. A smear result was judged to be negative if no parasites were seen after review of 100 highpower (400-fold magnification) fields. Final microscopy results were based on a rigorous quality control system, with re-reading of all blood smears by a second microscopist and resolution of any discrepancies by a third microscopist. The CD4+ T-cell counts were measured by a FACS Counter (Becton Dickinson, San Jose, CA, USA).

Measurement of serum IgG antibodies P. falciparum serum antibodies to peptides MSP3, HRP II and GLURP were measured by enzyme linked immunorsobent assay (ELISA) essentially as described before [23] using respective synthetic peptides as antigen and peroxidase-conjugated anti-human IgG or anti-human IgM

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as secondary antibody. Briefly, microtiter plate wells were coated with 0.5 to 1 microgram of peptide per well, incubated overnight at 4°C, and blocked with 5% skimmed milk for 2 hours at room temperature. Serum specimens were diluted 1:200, added in duplicates and incubated at room temperature for 1 h. Plates were washed 4 times between the coating and blocking steps. The microtiter plate wells were then incubated (45 min) with peroxidase-conjugated goat antihuman IgG (secondary antibody). Bound secondary antibody was quantified by developing colour with TMB (3, 3’, 5, 5’-Tetramethylbenzidine) substrate. Optical density (OD) was read at 450 nm with a reference at 620 nm using a plate reader. Sera of known unexposed individuals were used as controls. All the ELISAs for a given antigen were performed on the same day and on the same ELISA plate for day 0 and day 42 antibody tests of the same individual to eliminate the day to day variations. All samples with optical density readings above 1.4 were diluted further up to 1:1000 to ensure that ODs were less than 1.4. The OD obtained multiplied by the dilution factor. All specimens were analyzed twice or thrice in duplicates and the means of the ELISA OD used in the analysis. If the ELISA OD of an individual’s day 42 serum was higher than the same person’s corresponding value for the day 0 specimen, the increase was regarded as reflecting a positive antibody anamnestic response or boost. The ratio (D42 ELISA OD)/ (D0 ELISA OD) as well as % increase {(100 x (Day42 ELISA OD—Day0 ELISA OD)/Day0 ELISA OD} was then calculated.

Synthetic peptides Sero-reactivity to peptides of P. falciparum antigens was determined by ELISA using synthetic peptides which were coated onto ELISA microtiter plate wells. Amino acid sequences of the synthetic peptides [14, 22, 33, 34] representing P. falciparum asexual blood stage proteins were; GLURP: (NH2) CGDKNEKGQHEIVEVEEILPEGC (CONH2), HRP II: (NH2)GCAHHAADA HHAADAHHAADAHHAADGC(CONH2), and MSP3: (NH2) AKEASSYDYILGWEFGGGVPE HKKEEN (CONH2).

Statistical analysis and data presentation Data were analysed using STATA version 11.0 (Stata Corporation, College Station, Texas, USA) and Graph pad Prism 5. Data were tested for normality using Shapiro-Wilk W test. Differences between medians of IgG levels on day 0 and day 42 were evaluated by Mann-Whitney test while those among three or more groups were assessed by the non-parametric KruskalWallis test. Correlations were identified by Spearman rank test using actual or logarithmically transformed values P values 16 years

117 (32.3)

Level of education No education

109 (41.9)

Primary

111 (42.7)

Secondary

40 (15.4)

Cough on day 0 Yes

136 (37.6)

No

226 (62.4)

Septrin prophylaxis1 Yes

5 (16.6)

No

255(98.1)

Splenomegaly Palpable

60 (16.6)

Not palpable

302 (83.4)

1

denotes N = 260.

doi:10.1371/journal.pone.0124297.t001

General Characteristics In this study, 362 participants diagnosed with uncomplicated malaria were enrolled. About 58% (211/362) of the enrolled study participants were female (Table 1). Forty two percent (109/362) had not yet attended school for those aged less than five years or had no prior education training, 43% had attained primary level training while 15% had attained secondary level education. Among the study participants enrolled, nearly nineteen percent (68/362) were found to be HIV sero-positive. Of these HIV positive individuals, only seven percent (5/68) reported taking Cotrimoxazole prophylaxis therapy and none was on any anti retroviral therapy (ARV). 136 participants (38%) presented with symptoms of cough on day 0. Approximately 16% (57/362) of the study participants reported having taken an anti-helminthic treatment in the past one month before enrolling into this study. Only three percent (12/362) of the study participants reported that they had taken antimalarial treatment before coming to the health centre. Among the study participants, 67% (241/362) owned a mosquito net in their household and 61% (223/362) of the enrolled subjects slept under a mosquito net in the night preceding the day of enrolment (day 0). Only 38% (138/362) reported owning an insecticide treated bed net (ITN) while 8% (27/362) were not sure on whether their mosquito nets were treated. Enlargement of the spleen was observed in 17% (60/362) of the subjects. The median age of enrolled patients was 9.5 years (IQR: 3–21) and the reported duration of the malaria episode was 3 days (inter quartile range: 2–4). The median axillary temperature measured on day 0 was 37.95°C (IQR: 37.5–38°C).

Relationship between demographic characteristics and boosting after acute uncomplicated P. falciparum malaria We evaluated the association of all baseline factors and post-infection increase in parasite-specific IgG antibody among the study participants (Table 2). Sex of the study participants and co-

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Table 2. Multivariate analysis of subject characteristics and antibody boosting. Subject characteristic

Anti-GLURP IgG

Anti-HRPII

Anti-MSP3 IgG

Estimate1

P2

Estimate1

P2

Estimate1

P2

Less than 5 years

0

0

0

0

0

0

6–10 years

-0.17 (-0.46,0.12)

0.255

0.40 (-0.49,1.29)

0.367

0.28 (-0.12,0.68)

0.169

11–15 years

0.14 (0.50, 0.21)

0.437

0.57 (0.36, 1.49)

0.224

0.61 (0.13, 1.08)

0.013

>15 years

0.15 (-0.13, 0.43)

0.281

0.36 (-0.44, 1.15)

0.370

0.70 (0.31, 1.09)

0.001

Sex

-0.06 (-0.29, 0.16)

0.565

-0.22 (-0.81, 0.37)

0.460

0.12 (-0.20, 0.43)

0.467

HIV infection

-0.47 (-0.34, 0.24)

0.75

0.21 (-0.47, 0.90)

0.541

-0.30 (-0.69, 0.09)

0.137

Septrin prophylaxis

-0.28 (-0.58, 0.52)

0.92

0.77 (-1.56, 3.10)

0.510

-0.12 (-1.21, 0.96)

0.821

Duration of fever

0.04 (-0.02, 0.09)

0.163

-0.06 (-0.72, 0.47)

0.475

-0.07 (-0.15, 0.00)

0.064

Age group

Use of anti-malarial treatment before

-0.05 (-1.12, 0.17)

0.151

-0.69 (-2.43, 1.05)

0.431

-0.63 (-1.55, 0.29)

0.183

Use of deworming treatment

0.14 (-0.14, 0.43)

0.325

-0.07 (-0.86, 0.74)

0.862

-0.06 (-0.46, 0.33)

0.765

Owning a mosquito net in household

-0.22 (-0.46, 0.01)

0.06

0.22 (-0.37, 0.81)

0.465

-0.63 (-0.38, 0.25)

0.695

Sleeping under mosquito net in household

-0.22 (-0.25, 0.21)

0.874

0.27 (-0.32, 0.86)

0.367

0.08 (-0.23, 0.40)

0.580

Temperature

0.00 (-0.22, 0.22)

0.981

0.07 (-0.53, 0.66)

0.823

-0.12 (-0.43, 0.19)

0.435

Spleen

0.31 (0.03, 0.59)

0.030

0.84 (-0.05, 1.73)

0.063

0.42 (0.03, 0.80)

0.031

Malarial asexual parasitemia

0.07 (-0.01, 0.15)

0.108

-0.01 (-0.23, 0.21)

0.936

0.12 (0.08, 024)

0.036

1

denotes the coefficient/estimates.

2

denotes the p–value

doi:10.1371/journal.pone.0124297.t002

infection with HIV were not significantly associated with increase of antibody IgG against GLURP, HRPII and MSP3. Similarly, the duration of fever in days, use of antimalarial therapy before enrolment, self reported use of de-worming drugs, owning a mosquito net within the household and sleeping under a mosquito net in the night preceding the interview were not associated with post-infection increase in specific antibody IgG. However, the presence of a palpable spleen among the study participants was slightly associated with post-infection increase of IgG antibodies to GLURP and MSP3 (p = 0.030 and 0.031, respectively) while anti-HRPII antibodies were hardly affected by presence of palpable spleen.

Sero-prevalence to synthetic peptides on day 0 and age dependence of anti-peptide IgG on days 0 and 42 On day 0, the frequencies of IgG-sero-reactivity against GLURP, MSP3 and HRP II peptides were 75% (271/360), 60% (217/360) and 41% (148/358), respectively. On the other hand, the corresponding levels for IgM sero-prevalence to GLURP, HRP II and MSP3 were 80% (207/ 259), 42% (108/257) and 22% (57/256), respectively. For day 0 blood specimens, anti-peptide IgG levels were significantly associated with age for all the three peptides “Figs 1A, 1C, 1E and 1G”, the highest age group (16 years and above) showing the highest IgG levels. Likewise, for day 42, the median IgG ELISA units were higher in the eldest age group (> 16 years) studied. “Figs 1B, 1D, 1F and 1H”

Increase in serum anti-parasite IgG after a P. falciparum malaria attack For many subjects, there was a notable increase in the anti-peptide IgG levels from day 0 to day 42 for all the studied peptides GLURP, MSP3 and HRP II. However, the median anti-peptide

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Fig 1. Age dependent post-infection antibody response to P. falciparum synthetic peptides. To determine increase in parasite-specific IgG after P. falciparum malaria attack, sera collected at diagnosis (day 0) and 42 days later (day 42) were analyzed by ELISA. Graphs A, C, E and G represent antibody levels on day 0 while graphs B, D, F and H represent antibody levels on day 42. The level of serum anti-peptide IgG on day 0 was age dependent, participants older than 16 years showing the highest level of serum anti-peptide IgG. The Kruskal-Wallis test results: GLURP (H = 30.7, 2 df, P =